Alpha-helix mimetics and methods relating thereto

ABSTRACT

There are disclosed alpha-helix mimetics and methods relating to the same for imparting or stabilizing alpha-helicity to a peptide or protein. In one aspect, the alpha-helix mimetics contain six- or seven-membered rings covalently attached at the end or within the length of the peptide or protein. The alpha-helix mimetics render the resulting peptide or protein more stable with regard to thermal stability, as well as making the peptide or protein more resistant to proteolytic degredation. In addition, the alpha-helix mimetics may be used in standard peptide synthesis protocols.

STATEMENT OF GOVERNMENT INTEREST

This invention was made with government support under Grant No. GM-38260awarded by the National Institute of Health, and Grant No. CHE-8657046awarded by the National Science Foundation. The government may havecertain rights in this invention.

CROSS-REFERENCE TO RELATED APPLICATION

This application is a continuation of U.S. application Ser. No.08/079,316 filed Jun. 18, 1993, now abandoned.

TECHNICAL FIELD

This invention relates generally to alpha-helix mimetics and, morespecifically, to alpha-helix mimetics which stabilize the alpha-helicalstructure of a natural or synthetic peptide or protein.

BACKGROUND OF THE INVENTION

Proteins are polymers of amino acids in which the carbon atoms and amidegroups alternate to form a linear polypeptide, and with the amino acidside chains projecting from the α-carbon atom of each amino acid. Thesequence of amino acids and location of disulfide bridges (if any) areconsidered the "primary" protein structure. The "secondary" structure ofa protein refers to the steric relationship of amino acid residues thatare in close proximity to one another in the linear sequence. Suchsteric relationships give rise to periodic structure, including thealpha-helix.

The alpha-helix is a rod-like structure wherein the polypeptide chainforms the inner part of the rod, and the side chains extend outward in ahelical array. The alpha-helix is stabilized by hydrogen bonds betweenthe NH and CO groups of the polypeptide chain. More specifically, thehydrogen of the NH group of each amino acid (i.e., amino acid residue"n") is hydrogen bonded to the oxygen of the CO group that is locatedfour amino acid residues behind in the linear polypeptide (i.e., aminoacid residue "n-4"). Such hydrogen bonding is illustrated below:##STR1## While only a single hydrogen bond is depicted above for purposeof illustration, each of the CO and NH groups of the linear polypeptideare hydrogen bonded in the alpha-helix. In particular, each amino acidis related to the next by a translation of 1.5 Å along the helix axisand a rotation of 100°, which gives 3.6 amino acid residues per turn ofthe alpha-helix. The pitch of the alpha-helix is 5.4 Å (the product ofthe translation, 1.5 Å, and the number of residues per turn, 3.6), andthe diameter of the alpha-helix is 2.3 Å. The "screw sense" of thealpha-helix can be right-handed (clockwise) or left-handed(counter-clockwise). While a few left-handed alpha-helixes do exist,most alpha-helixes found in naturally occurring proteins areright-handed.

In the absence of interactions other than hydrogen-bonding, thealpha-helix is the preferred form of the polypeptide chain since, inthis structure, all amino acids are in identical orientation and eachforms the same hydrogen bonds. Thus, polyalanine (i.e.,[--NHCH(CH₃)CO--]n) exists as an alpha-helix. However, the presence ofother amino acids within the polypeptide chain may cause instability tothe alpha-helix. In other words, the amino acid side chains do notparticipate in forming the alpha-helix, and may hinder or even preventalpha-helix formation. A striking example of such side chain dependencyon alpha-helix formation is polylysine (i.e., [--NHCH((CH₂)₄NH₂)CO--]n). At a pH below 10, the NH₂ moiety in the side chain oflysine is charged (i.e., NH₃ ⁺), and electrostatic repulsion totallydestroys the alpha-helix structure. Conversely, at a pH above 10, thealpha-helix structure is preferred.

The alpha-helix constitutes one of the principle architectural featuresof peptides and proteins, and are important structural elements in anumber of biological recognition events, including ligand-receptorinteractions, protein-DNA interactions, protein-RNA interactions, andprotein-membrane interactions. In view of the important biological roleplayed by the alpha-helix, there is a need in the art for compoundswhich can stabilize the intrinsic alpha-helix structure. There is also aneed in the art for methods of making stable alpha-helix structures, aswell as the use of such stabilized structures to effect or modifybiological recognition events which involve alpha-helical structures.The present invention fulfills these needs and provides further relatedadvantages.

SUMMARY OF INVENTION

Briefly stated, the present invention is directed to alpha-helixmimetics and, more specifically to alpha-helix mimetics which stabilizethe alpha-helical structure of a natural or synthetic peptide orprotein.

In one aspect of this invention, alpha-helix mimetics are disclosedhaving the following structures I and II: ##STR2## wherein Z is anoptional methylene moiety (i.e., --CH₂ --); R¹ -R⁵ are each selectedfrom amino acid side chain moieties or derivatives thereof; and X and Yrepresent the remainder of the molecule. Amino acid side chain moietiesinclude, but are not limited to, the amino acid side chains of glycine,alanine, valine, leucine, isoleucine, lysine, arginine, histidine,aspartic acid, glutamic acid, asparagine, glutamine, phenylalanine,tyrosine, tryptophan, cysteine, methionine, serine and threonine.Derivatives of amino acid side chain moieties include, but are notlimited to, straight chain or branched, cyclic or noncyclic, substitutedor unsubstituted, saturated or unsaturated lower chain alkyl and lowerchain aryl moieties.

In another aspect, an alpha-helix modified peptide or protein isdisclosed wherein an alpha-helix mimetic of this invention is covalentlyattached to at least one amino acid of a peptide or protein. In thisembodiment, X and/or Y in the above structures I and II represent one ormore amino acids of the peptide or protein. In still a further aspect, amethod for imparting and/or stabilizing alpha-helicity of a peptide orprotein is disclosed. This method includes covalently attaching one ormore alpha-helix mimetics of this invention within, or to the end of, apeptide or protein.

Other aspects of this invention will become apparent upon reference tothe attached figures and the following detailed description.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a schematic illustrating the synthesis of representativealpha-helix mimetics of this invention.

FIGS. 2(A), 2(B) and 2(C) are further schematics illustrating thesynthesis of representative alpha-helix mimetics of this invention.

DETAILED DESCRIPTION OF THE INVENTION

As mentioned above, the alpha-helix is an important structural componentfor many biological recognition events. The alpha-helix mimetics of thisinvention serve to impart and/or stabilize the alpha-helical structureof a natural or synthetic peptide or protein, particularly with regardto thermal stability. In addition, the alpha-helix mimetics of thisinventionare more resistant to proteolytic breakdown, thus rendering apeptide or protein containing the same more resistant to degradation.

The alpha-helix mimetics of this invention have the following structuresI and II: ##STR3##wherein Z (structure I) is an optional methylenemoiety (i.e., --CH₂ --); R¹ -R⁵ (structure I) and R¹ -R⁴ (structure II)are each selected from amino acid side chain moieties or derivativesthereof; and X and Y represent the remainder of the molecule. Althoughstructures I and II have been depicted as preferred embodiments forright-handed alpha-helices, one skilled in the art will recognize thatthemirror images of structures I and II would be used in left-handedalpha-helices.

When the optional methylene moiety Z is present in structure I, thealpha-helix mimetic is a seven-membered ring having the followingstructure III: ##STR4##wherein R¹ -R⁵ are each selected from amino acidside chain moieties or derivatives thereof; and X and Y represent theremainder of the molecule. On the other hand, when the optionalmethylene moiety Z of structure I is not present, the alpha-helixmimetic is a six-membered ringhaving the following structure IV:##STR5##wherein R¹ -R⁵ are each selected from amino acid side chainmoieties or derivatives thereof; and X and Y represent the remainder ofthe molecule.

The term "remainder of the molecule", as represented by X and Y in theabove structures, may be any chemical moiety. For example, when thealpha-helix mimetic is located within the length of a peptide orprotein, X and Y may represent amino acids of the peptide or protein.Alternatively, if two or more alpha-helix mimetics are linked, the Ymoiety of a first alpha-helix mimetic may represent a second alpha-helixmimetic while, conversely, the X moiety of the second alpha-helixmimetic represents the first alpha-helix mimetic. When the alpha-helixmimetic is located at the end of a peptide or protein, or when thealpha-helix mimetic is not associated with a peptide or protein, Xand/or Y may represent a suitable terminating moiety. Such terminatingmoieties include, but are not limited to, --H, --OH, --OR, --NH₂, --CHO,--NHRand --COR (where R is a lower chain alkyl or aryl moiety), as wellas suitable peptide synthesis protecting groups (such as BOC and FMOC).

As used herein, the term "an amino acid side chain moiety" representsany amino acid side chain moiety present in naturally occurringproteins, including (but not limited to) the naturally occurring aminoacid side chain moieties identified in Table 1.

                  TABLE 1                                                         ______________________________________                                        Amino Acid Side Chain Moiety                                                                         Amino Acid                                             ______________________________________                                        H                      Glycine                                                CH.sub.3               Alanine                                                CH(CH.sub.3).sub.2     Valine                                                 CH.sub.2 CH(CH.sub.3).sub.2                                                                          Leucine                                                CH(CH.sub.3)CH.sub.2 CH.sub.3                                                                        Isoleucine                                             (CH.sub.2).sub.4 NH.sub.3.sup.+                                                                      Lysine                                                 (CH.sub.2).sub.3 NHC(NH.sub.2)NH.sub.2.sup.+                                                         Arginie                                                 ##STR6##              Histidine                                              CH.sub.2 COO.sup.-     Aspartic acid                                          CH.sub.2 CH.sub.2 COO.sup.-                                                                          Glutamic acid                                          CH.sub.2 CONH.sub.2    Asparagine                                             CH.sub.2 CH.sub.2 CONH.sub.2                                                                         GLutamine                                               ##STR7##              Phenylalanine                                           ##STR8##              Tyrosine                                                ##STR9##              Tryptophan                                             CH.sub. 2 SH           Cysteine                                               CH.sub.2 CH.sub.2 SCH.sub.3                                                                          Methionine                                             CH.sub.2 OH            Serine                                                 CH(OH)CH.sub.3         Threonine                                              ______________________________________                                    

Other naturally occurring side chain moieties of this invention include(but are not limited to) the side chain moieties of 3,5-dibromotyrosine,3,5-diiodotyrosine, hydroxylysine, γ-carboxyglutamate, phosphotyrosine,phosphoserine and glycosylated amino acids such as glycosylated serineand threonine.

In addition to naturally occurring amino acid side chain moieties, theamino acid side chain moieties of the present invention also includevarious derivatives thereof. As used herein, a "derivative" of an aminoacid side chain moiety includes all modifications and/or variations tonaturally occurring amino acid side chain moieties. For example, theaminoacid side chain moieties of alanine, valine, leucine, isoleucineand phenylalanine may generally be classified as lower chain alkyl oraryl moieties. Derivatives of amino acid side chain moieties includeother straight chain or branched, cyclic or noncyclic, substituted orunsubstituted, saturated or unsaturated lower chain alkyl or arylmoieties. As used herein, "lower chain alkyl moieties" may contain from1-12 carbon atoms, and "lower chain aryl moieties" may contain from 6-12carbon atoms. Substituted derivatives of lower chain alkyl or arylmoieties of this invention include (but are not limited to) one or moreofthe following chemical moieties: --OH, --OR, --COOH, --COOR, --CONH₂,--NH₂, --NHR, --NRR, --SH, --SR and halogen (including F, Cl, Br and I),wherein R is a lower chain alkyl or aryl moiety. Moreover, cyclic lowerchain alkyl and aryl moieties of this invention include naphthalene,aswell as heterocyclic compounds such as thiophene, pyrrole, furan,imidazole, oxazole, thiazole, pyrazole, 3-pyrroline, pyrrolidine,pyridine, pyrimidine, purine, quinoline, isoquinoline and carbazole.

As mentioned above, the alpha-helix mimetics of this invention serve toimpart and/or stabilize the alpha-helicity of a protein or peptide. Thealpha-helix mimetic may be positioned at either the C-terminus orN-terminus of the protein or peptide, or it may be located within theprotein or peptide itself. In addition, more than one alpha-helixmimetic of the present invention may be incorporated in a protein orpeptide.

The alpha-helix mimetics of this invention have broad utility in avariety of naturally occurring or synthetic peptides and proteins. Forexample, neuropeptide Y ("NPY"), a polypeptide amide of 36 amino acids,is a potentvasoconstrictor and neuromodulator, and antagonists to NPYhave anti-hypertensive activity. C-terminal analogs of NPY havepreviously beenconstructed, including the following analogs:Ac--RAAANLITRQRY--NH₂ and Ac--RAAANAAARQRY--NH₂ (Ac represents that theamino-terminus is acetylated, and --NH₂ indicates that thecarbon-terminus is an amide). The biological activity of the aboveanalogs, as evidenced by binding to the NPY binding site, has beencorrelated to its alpha-helicity(see, Jung et al., Biopolymers31:613-19, 1991). By substituting one or more of the alpha-helixmimetics of this invention for select amino acids within an NPY analogpolypeptide, the alpha-helicity of the NPY analog maybe enhanced, thusincreasing its binding affinity and improving its biological activity.Suitable assays for determining bioresponse and binding are known,including the Guinea Pig Atrial Contraction Assay (Panlabs Inc.,Bothell, WA; see also, Giuliani et al., Br. J. Pharmacol. 98:407-412,1989), Discoveryscreen™ Neuropeptide Y Binding (Panlabs, Inc., Bothell,WA; see also, Walker et al., Molec. Pharmacol. 34:778-792; Saria et al.,Eur. J. Pharmacol. 107:105-107, 1985), Rat Vas Deferens Relaxation (EFS)(Panlabs, Inc., Bothell, WA; see also, Wahlested et al., RegulatoryPeptide 13:307-318, 1986; Martel et al., J. Pharmacol. Exp. Ther.38:494-502, 1990)(which references are hereby incorporated by referencein their entirety).

For example, the following structure V may be made by substituting analpha-helix mimetic of structure III above for the "AAA" sequence of theNPY analog Ac--RAAANLITRQRY--NH₂ : ##STR10##Similarly, the followingstructure VI may be made by substituting an alpha-helix mimetic ofstructure IV above for the "AAA" sequence of the NPY analogAc--RAAANLITRQRY--NH₂ : ##STR11##Alternatively, more than onealpha-helix mimetic of the present invention may be utilized. Forexample, the following structure VII may be made by substituting twoalpha-helix mimetics of structure III for the "AAANAAA" sequence of theNPY analog Ac--RAAANAAARQRY--NH₂ : ##STR12##

A further example of the utility of the alpha-helix mimetics of thepresentinvention may be illustrated with regard to cytokines. Manycytokines (e.g., hGH, IL2 and IL4) are organized into a bundle ofalpha-helical structures. In many instances the C-terminal alpha-helixis a critical site of interaction with its corresponding receptor. Forexample, C-terminal-stabilized alpha-helical peptides (stabilized withone or more of the alpha-helix mimetics of this invention) may serve asantagonists ofIL2 and IL4, and have utility as anti-inflammatory agents.More specifically, the C-terminal of both IL2 and IL4 have been foundcritical in ligand-receptor interaction (see, Landgraf et al., JBC264:816-22, 1989and Ramanathan et al., Biochemistry 32:3549-56, 1993).The respective sequences for these regions are: .sup.(119)NRWITFCQSIISTLT.sup.(133) and .sup.(111) NFLERLKTIMREKYSPCSS.sup.(129).By synthesizing a peptide which contains one or more of the alpha-helixmimetics of this invention in place of certain amino acids in the abovesequences, peptides can be made which will mimic the C-terminal of IL2or IL4, thus serving as IL 2 or IL4receptor antagonists. For example,the following structures VIII, IX and X can be made by substituting oneor more of the alpha-helix mimetics of structure III for select aminoacids with the above IL2 C-terminal sequence: ##STR13##(In structureVIII the cysteine residue of the IL2 C-terminal sequence has beenreplaced with alanine (i.e., C→A) since cysteine is prone to oxidation,and isoleucine has been changed to leucine (i.e., I→L) in structuresVIII, IX and X to increase alpha-helicity.)

While the utility of the alpha-helix mimetics of this invention havebeen disclosed with regard to certain embodiments, it will be understoodthat awide variety and type of compounds can be made which includes thealpha-helix mimetics of the present invention. For example, analpha-helixof this invention may be substituted for two or more aminoacids of a peptide or protein. In addition to improving and/or modifyingthe alpha-helicity of a peptide or protein, especially with regard tothermal stability, the alpha-helix mimetics of this invention also serveto inhibit proteolytic breakdown. This results in the added advantage ofpeptides or proteins which are less prone to proteolytic breakdown dueto incorporation of the alpha-helix mimetics of this invention.

The alpha-helix mimetics of this invention may generally be synthesizedby the condensation of a suitable derivative of a carboxylic acid XI orXII with a hydrazine moiety XIII: ##STR14##wherein Z (structure XI) isan optional methylene moiety (i.e., --CH₂ --); R³ -R⁵ (structure XI), R³-R⁴ (structure XII) andR¹ -R² (structure XIII) are each selected fromamino acid side chain moieties or derivatives thereof; P'-P"" are eachselected from suitable peptide synthesis protecting groups, X¹ is asuitable carboxylic acid protecting group (such as methyl or benzyl),and X² is a suitable leaving group (such as chloride or imidazole).Following condensation of structures XI or XII with structure XIII, theresulting compound may then be cyclized to yield the correspondingstructure I or IIabove. A more detailed disclosure with respect tosynthesis of the alpha-helix mimetics of this invention is presented inthe examples below.

The alpha-helix mimetics of the present invention may be used instandard peptide synthesis protocols, including automated solid phasepeptide synthesis. Peptide synthesis is a stepwise process where apeptide is formed by elongation of the peptide chain through thestepwise addition ofsingle amino acids. Amino acids are linked to thepeptide chain through theformation of a peptide (amide) bond. Thepeptide link is formed by couplingthe amino group of the peptide to thecarboxylic acid group of the amino acid. The peptide is thus synthesizedfrom the carboxyl terminus to the amino terminus. The individual stepsof amino acid addition are repeated until a peptide (or protein) ofdesired length and amino acid sequence is synthesized.

To accomplish peptide (or protein) synthesis as described above, theamino group of the amino acid to be added to the peptide should notinterfere with peptide bond formation between the amino acid and thepeptide (i.e., the coupling of the amino acid's carboxyl group to theamino group of the peptide). To prevent such interference, the aminogroup of the amino acidsused in peptide synthesis are protected withsuitable protecting groups. Typical amino protecting groups include BOCand FMOC groups. Accordingly, in one embodiment of the presentinvention, the alpha-helix mimetics of the present invention bear a freecarboxylic acid group and a protected amino group, and are thus suitablefor incorporation into a peptide by standard synthetic techniques.

The following examples are offered by way of illustration, notlimitation.

EXAMPLES Example 1 Synthesis of Representative Alpha-Helix Mimetics ofStructure I

A. Synthesis of Structure XIV:

This example illustrates the synthesis an alpha-helix mimetic having thefollowing structure XIV: ##STR15##

The following synthesis is presented schematically in FIG. 1. Synthesisof structure XIV was generally accomplished by condensation of hydrazinederivative (3) with carboxylic acid derivative (5). More specifically,protected hydrazine (3) was prepared in three steps from ethylisobutyrate. First, the lithium enolate of the ethyl ester (generated bytreatment with lithium diisopropylamide) was added to di-t-butylazodicarboxylate to produce (after ammonium chloride quench) additionproduct (1) in 85 percent yield. The t-butyl carboxylate groups of (1)were removed by acid hydrolysis in ethyl acetate saturated withhydrochloric acid and the resulting hydrazine salt treated withbenzylchloroformate, triethylamine, and dimethylaminopyridine inmethylenechloride. N-benzylcarbamate (2) was thus produced in 59 percentyield. Protected hydrazine (3) was prepared in 99 percent yield byreaction of N-benzylcarbamate (2) with an excess oftrimethylsilylchloride in triethylamine. Protected hydrazine (3)prepared as described was suitable for condensation.

A protected version of glutamic acid (4) (i.e., amino groupt-butylcarbonate protected and side chain carboxylic acid benzylprotected) (Advanced Chemtech, Louisville, Ky.) was the startingmaterial for the synthesis of the carboxylic acid. Specifically,carboxylic acid (4) was carboxy activated by conversion to thecorresponding acid fluoride(5) by reaction with cyanuric fluoride andpyridine in methylene chloride. Acid fluoride (5) is a reactiveintermediate and was not isolated but rather treated directly withprotected hydrazine (3) and silver cyanide (2equivalents) in benzene toproduce diester (6) in 80% yield. Hydrogenolysisof (6) over 10 percentpalladium on carbon in ethanol removed both benzyl and benzyl carbamoylprotecting groups to generate intermediate (7) which was self-condensedto the corresponding cyclic hydrazide in 52 percent yield by treatmentwith 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride and1-hydroxy-benzotriazole hydrate in aqueous tetrahydrofuran. Esterhydrolysis of the cyclic hydrazide with ethanolic potassium hydroxidegave alpha-helix mimetic XIV in 88 percent yield. The characteristics ofthe ¹ H-NMR spectrum of the methyl ester of structure XIV were asfollows: BOC--NH (5.45δ, 1H, broad s), αCH (4.37δ, 1H, ddd, J₁ =7Hz, J₂=5.7Hz, J₃ =4.8 Hz), OCH₃ (3.72δ, 3H, s), --CH₂ --CO--(2,78δ, 2H, m ),--CH₂ --CH₂ --CO-- (1.85δ, 2H, m), BOC--NH (1.42δ, 9H, s), --C(CH₃)₂(1.32δ, 6H, s). Similarly,the characteristics of the ¹³ C--NMR spectrumof the methyl ester of structure XVI were as follows: --CO-- (175 δ,172δ, 171δ, 156.5δ), --NC(CH₃)₂ CO-- (82δ), --N--CH--CO-- (62.5δ),--OCH₃ (53δ), --CH₂ CO-- (31.5δ), --CH₂ CH₂ CO-- (29.0δ), --NC(CH₃)₂CO--(25.5δ, 24.0δ ).

B. Synthesis of Structure XV:

This example illustrates the synthesis of an alpha-helix mimetic havingthefollowing structure XV: ##STR16##

The alpha-helix of structure XV is prepared in a synthetic reactionscheme identical to the scheme disclosed in FIG. 1 for structure XIV,except thatinstead of using a protected version of glutamic acid (i.e.,compound (4) in FIG. 1) as a starting material, the following protectedversion of aspartic acid (4') was employed: ##STR17##C. Synthesis ofOther Alpha-Helix Mimetics

Other synthetic routes may be utilized to prepare alpha-helix mimeticsof structure I. For example, to prepare derivatives of structure I whereR³ is methyl, α-methyl glutamic or aspartic acid may be utilized in areaction sequence as illustrate in FIG. 1. These α-amethyl derivativesmay be prepared as described in Zydowsky et al. (J. Org. Chem. 55:5437,1990). In this reaction sequence, the methyl group is derived fromreactant alanine. 2-hydroxybenzaldehyde is condensedwith alanine underbasic conditions to yield an intermediate which may be alkylated witheither a 2-bromopropionic acid equivalent (or a 2-bromoethanoic acidequivalent) to yield, after hydrolysis, a α-methyl-glutaric (oraspartic) acid derivative suitable for condensation with a suitablehydrazine to provide the alpha-helix mimetic of structure I.

Alternatively, R⁴ or R⁵ substituted derivatives of structure I may beprepared as described in FIG. 1 utilizing 3-substituted glutamicoraspartic acid derivatives which may be prepared according to theprocedure described in Baldwin et al. (Tetrahedron 45:1465, 1989). Inthis procedure, protected versions of these amino acids may be directlyalkylated at the carbon alpha to the side chain carbonyl. Utilization ofthe resulting substituted derivatives in the reaction sequence describedillustrated in FIG. 1 provides derivatives of structure I substituted atR⁴ or R⁵.

In addition to variations of the carboxylic acid derivatives utilized inthe synthesis of alpha-helix mimetics of structure I, varioussubstituted hydrazines may also be utilized. For example, hydrazineformation from acylcarbamates as described in Gennari et al. (J. Amer.Chem. Soc. 108:6394, 1986) may be utilized to provide substitutedhydrazines which, when submitted to the reaction conditions described inFIG. 1, will provide derivatives of structure I with appropriatesubstitution variations at positions R¹ and R².

Example 2 Synthesis of Representative Alpha-Helix Mimetics of StructureII

This example illustrates the synthesis of an alpha-helix mimetic havingthefollowing structure XVI: ##STR18##Synthesis of structure XVI may beaccomplished by two methods. The first method is the two-stepcondensation of a protected hydrazine with a suitable derivative of acarboxylic acid as generally described in Example1. In this method, thepreparation of the carboxylic acid derivative is representedschematically in FIG. 2(A). The second method is represented in FIG.2(B), and utilizes the protected hydrazine of Example 1.

A. Synthesis of Structure XVI:

To prepare an alpha-helix mimetic of structure XVI by the two-stepcondensation method of Example 1, a suitable derivative of carboxylicacidXII is first prepared. Referring to FIG. 2(A), such a derivative(wherein R³ and R⁴ are hydrogen and X² is imidazole) may be synthesizedin three steps starting with a protected version of serine (8)(i.e.,amino group t-butylcarbonate protected) (Advanced Chemtech, Louisville,Ky). Specifically, protected serine (8) is treated with diethylazodicarboxylate, triphenylphosphine, and hydrogen azide to yield anintermediate serine azide derivative. Hydrogenolysis of the intermediateazide over palladium on carbon yields amine (9), a serine derivativewhere the side chain hydroxy has been substituted with an aminogroup.Alternatively, amine (9) may be prepared from aspartic acid via Curtiusrearrangement. Treatment of amine (9) with carbonyl diimidazole producescarboxylic acid derivative (10) (i.e., structure XII where R³ and R⁴ arehydrogen, P" is BOC, and X² is imidazole). This carboxylic acidderivative is suitable for condensation with protected hydrazine (3) ofFIG. 1 to yield structure XVI in a reaction sequence as described inExample 1.

B. Alternative Synthesis of Structure XVI:

Referring to FIG. 2(B), an alpha-helix mimetic having structure XVI maybe prepared by cyclization of a suitable diamine. In this method, theseven-membered ring of the alpha-helix mimetic is formed in the lastsynthetic step by treating a suitable diamine with carbonyl diimidazole.The reaction sequence begins with a protected version of serine (8)(i.e.,amino group t-butylcarbonate protected and carboxyl group methylester protected) (Advanced Chemtech, Louisville, Ky). As with the methodillustrated in FIG. 2(A), the side chain hydroxyl of the serine methylester is first converted to an azide group with diethylazodicarboxylate, triphenylphosphine, and hydrogen azide. The serinemethyl ester is then hydrolyzed to the corresponding carboxylic acid(11) and then converted toits acid fluoride by treatment with cyanuricfluoride. The intermediate acid fluoride is then treated with protectedhydrazine (3) of FIG. 1 to yield azide (12). Hydrogenolysis of (12) overpalladium on carbon reduces the azide group and removes the benzylcarbamoyl group to produce diamine (13). The seven-membered ring of thealpha-helix mimetic is formed by treatment of diamine (13) with carbonyldiimidazole and dimethylaminopyrridine, resulting in ethyl ester (14).Hydrolysis of (14) yields the alpha-helix mimetic of structure XVI.

Alternatively, referring to FIG. 2(C), diamine (13) may be prepared fromBOC-aspartic acid-α-methyl ester (15) via Curtius rearrangement.Treatment of (15) with diphenyl-phosphoryl azide, followed by reactionwith benzyl alcohol, yields an intermediate benzyl carbamoyl ester whichmay be hydrolyzed to the corresponding carboxylic acid (16). Reaction of(16) with protected hydrazine (3) as shown in FIG. 2(B) producesprotecteddiamine (17) which, upon hydrogenolysis, provides diamine (13).

Example 3 Synthesis of a Representative Peptide Containing anAlpha-Helix Mimetic

This example illustrates the synthesis of a peptide containing analpha-helix mimetic of the present invention having structure VI.

Specifically, an alpha-helix mimetic having structure XV was synthesizedaccording to the disclosure of Example 1, and incorporated into apeptide by standard solid phase peptide synthetic techniques to yieldstructure VI. The peptide of structure VI was synthesized on either PAMor MBHA (P-methyl benzhydrylamine) resin using an Advanced Chemtech ACT90 model synthesizer. Couplings were conducted using1-hydroxybenzotriazole, benzotriazolyl-oxy-tris-(dimethylamino)phosphonium hexafluorophosphate, and diisopropylethylamine in a mixtureof dichloromethane and dimethylformamide at room temperature. Cleavagefrom the resin was accomplished with either hydrogen fluoride/anisole orammonolysis. The resin was washed with ether and extracted with diluteacetic acid and evaporated to dryness in vacuo. The crude residue waspurified by reverse phase C18 high performance liquid chromatography.

Example 4 Alpha-Helicity of a Representative Peptide Containing anAlpha-Helix Mimetic

This example illustrates the enhanced alpha-helicity of a peptidecontaining an alpha-helix mimetic of the present invention.

Alpha-helicity of a peptide is typically determined by measuring itscircular dichroism (CD), and CD data is normally presented as meanresidueellipticies [Θ]_(m). Alpha-helical peptides show two negativeCotton effects at 208 nm and 222 nm, and a positive Cotton effect at 193nm, while the CD spectra of peptides with random coil secondarystructure are dominated by the increasing negative Cotton effect atshorter wavelength. Alpha-helicity may be estimated from the value at222 nm, and by comparing the negative Cotton effects at 222 nm and 208nm. Increasing fraction of [Θ]_(m) (222 nm) divided by [Θ]_(m) (208 nm)correlates with increasing alpha-helicity. High values for [Θ]_(m)(208nm) compared to [Θ]_(m) (222 nm) and a shifting minimum from 208 nm toshorter wavelengths indicate random coil conformation.

An NPY analog having the structure Ac--RAAANLITRQRY--NH₂ was synthesizedaccording to standard solid phase peptide synthetic techniques. The NPYanalog was then dissolved in water at a concentration of 75 μM, and itsCD measured using a JASCO J500 CD spectrometer at a temperature of both3° C. and 25° C. Structure VI was synthesized according to Example 3,and its CD was similarly measured at both 3° C. and 25° C. (at aconcentration of 75 μM). The results of this experiment are presented inTable 2.

                  TABLE 2                                                         ______________________________________                                                    Circular Dichroism                                                Compound      222/208 3° C.                                                                     222/208 25° C.                                ______________________________________                                        NPY Analog    0.38       0.41                                                 Structure VI  0.64       0.66                                                 ______________________________________                                    

As illustrated by the data in Table 2, the alpha-helicity of the NPYanalogwas enhanced by the substitution of an alpha-helix mimetic of thisinvention within the peptide sequence. In particular, at 3° C.alphahelicity was increased from 0.38 to 0.64, and at 25° C. itwasincreased from 0.41 to 0.66. Since biological activity of the NPYanalog has been correlated to alpha-helicity, the ability of thealpha-helix mimetics of this invention to enhance alpha-helicity willcorrespondingly enhance the biological activity of the NPY analogcontaining the alpha-helix mimetic.

From the foregoing, it will be appreciated that, although specificembodiments of this invention have been described herein for purposes ofillustration, various modifications may be made without deviating fromthespirit and scope of the invention. Accordingly, the invention is notlimited except by the appended claims.

    __________________________________________________________________________    SEQUENCE LISTING                                                              (1) GENERAL INFORMATION:                                                      (iii) NUMBER OF SEQUENCES: 9                                                  (2) INFORMATION FOR SEQ ID NO:1:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 12 amino acids                                                    (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: peptide                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:1:                                       ArgAlaAlaAlaAsn LeuIleThrArgGlnArgTyr                                         1510                                                                          (2) INFORMATION FOR SEQ ID NO:2:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 12 amino acids                                                    (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: peptide                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:2:                                       ArgAlaAla AlaAsnAlaAlaAlaArgGlnArgTyr                                         1510                                                                          (2) INFORMATION FOR SEQ ID NO:3:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 8 amino acids                                                     (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: peptide                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:3:                                       As nLeuIleThrArgGlnArgTyr                                                     15                                                                            (2) INFORMATION FOR SEQ ID NO:4:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 7 amino acids                                                     (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: peptide                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:4:                                       AlaAlaAlaAsnAlaAlaAla                                                          15                                                                           (2) INFORMATION FOR SEQ ID NO:5:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 4 amino acids                                                     (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: peptide                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:5:                                       ArgGlnArgTyr                                                                  (2) INFORMATION FOR SEQ ID NO:6:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A ) LENGTH: 15 amino acids                                                   (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: peptide                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:6:                                       AsnArgTrpIleThrPheCysGlnSerIleIleSerThrLeuThr                                 1510 15                                                                       (2) INFORMATION FOR SEQ ID NO:7:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 19 amino acids                                                    (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: peptide                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:7:                                       AsnPheLeuGluArgLeuLysThrIleMetArgGluLysTyrSerPro                              1 51015                                                                       CysSerSer                                                                     (2) INFORMATION FOR SEQ ID NO:8:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 12 amino acids                                                    (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: peptide                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:8:                                       IleThrPheAlaG lnSerLeuLeuSerThrLeuThr                                         1510                                                                          (2) INFORMATION FOR SEQ ID NO:9:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 8 amino acids                                                     (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: peptide                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:9:                                       GlnSer LeuLeuSerThrLeuThr                                                     15                                                                        

I claim:
 1. An alpha-helix mimetic having the structure: ##STR19##wherein Z is an optional methylene moiety; R¹ through R⁵ are eachselected from hydrogen and methyl; and X and Y are selected from thegroup consisting of amino acids, alpha-helix mimetics and terminatingmoleties.
 2. The alpha-helix mimetic of claim 1 having the structure:##STR20##
 3. The alpha-helix mimetic of claim 1 having the structure:##STR21##
 4. An alpha-helix mimetic having the structure: ##STR22##wherein R¹ through R⁴ are each selected from hydrogen and methyl; and Xand Y are selected from the group consisting of amino acids, alpha-helixmimetics and terminating moieties.
 5. An alpha-helix modified peptide orprotein, comprising a peptide or protein and an alpha-helix mimetic, thealpha-helix milnetic being covalently attached to at least one aminoacid of the peptide or protein, wherein the alpha-helix mimetic has thestructure: ##STR23## wherein Z is an optional methylene moiety; R¹through R⁵ are each selected from hydrogen and methyl; and at least X orY is an amino acid of the peptide or protein.
 6. The alpha-helixmodified peptide or protein of claim 5 wherein both X and Y are amineacids of the peptide or protein.
 7. An alpha-helix modified peptide orprotein, comprising a peptide or protein and an alpha-helix mimetic, thealpha-helix mimetic being covalently attached to at least one amino acidof the peptide or protein, wherein the alpha-helix mimetic has thestructure: ##STR24## wherein R¹ through R⁴ are each selected fromhydrogen and methyl; and at least X or Y is an amino acid of the peptideor protein.
 8. The alpha-helix modified peptide or protein of claim 7wherein both X and Y are amino acid of the peptide or protein.